It is generally known that free gas microbubbles with an interfacial tension are prone to dissolution, even in gas saturated solution as a consequence of the Laplace pressure across the gas-liquid interface. The Laplace pressure is believed to act as a driving force for the diffusion of gas from the bubble to the surrounding environment (see Epstein et al. J. Chem. Phys. 18(11): 1505-1509 (1950)). Stabilization of microbubbles against such dissolution may be achieved by the creation of a shell, typically composed of either a protein (e.g., denatured albumin) or a surfactant such as phospholipid, present at the surface of the bubble (see, Fox et al., The Journal of the Acoustical Society of America, 26(6): 984-989 (1954); Strasberg, M. Journal of the Acoustical Society of America 31(2): 163-176 (1959); and Manley, British Journal of Applied Physics 11(January): 38-42 (1960)). Protein-coated contrast agents that are air-filled (such as, for example, Albunex.TM. sold by Molecular Biosystems, San Diego and Mallinckrodt, St. Louis) or perfluorocarbon-filled (such as, for example, Optison.TM. sold by Molecular Biosystems, San Diego and Mallinckrodt, St. Louis) are currently approved for medical use.
Notwithstanding any potential advantages of albumin materials, the use of phospholipid may be very desirable. More particularly, the amphiphilicity of the lipid molecule may cause it to spontaneously order itself at an air-water interface in such a way that the hydrophilic headgroup region of the lipid is exposed to the aqueous phase, while the hydrophobic tail region is oriented toward the air (see Tanford, C. The Hydrophobic Effect: Formation of Micelles and Biological Membranes. New York. John Wiley & Sons, Inc.1980). Therefore, phospholipid tends to self-assemble and form a monolayer shell at the surface of a gas bubble, under appropriate temperature and salt concentration conditions. In contrast to phospholipid, denatured albumin tends to form unoriented multilayers.
In addition, a wealth of data on the properties and structure of phospholipid has been established (see Marsh, D. CRC Handbook of Lipid Bilayers. Boca Raton, CRC Press (1990)). As an example, the temperature-dependent phase behavior (transition between liquid crystalline and gel states) of the phospholipid materials used to create shells is typically defined in bilayers and, to some extent, in monolayers as well. Conversely, in a fused, denatured shell, albumin is believed to have no phase transition.
Advantageously, phospholipid may be readily derivatized with fluorescent, polymeric, or binding moieties via chemical modification. Coated microbubbles can thus be tagged with dye markers for structural studies, engineered to potentially avoid uptake and extravasation in the body. The coated microbubble can be targeted to specific in vivo sites. These chemical groups, attached to lipid headgroups, tend to be precisely oriented at the surface of the shell. Not being bound by any theory, the orientation is believed to be possibly caused by the spontaneous arrangement of lipid at the bubble surface such that all headgroups are oriented toward the external aqueous medium. Albumin shells are believed to differ in that their preferential orientation of surface derivatized groups is difficult if not impossible to achieve.
Furthermore, phospholipid shells on microbubbles are capable of accommodating surface area changes of at least 40 percent for condensed monolayers. Such accommodation may be due to the ability of phospholipid monolayers to expand through multiple phase transitions (i.e., liquid condensed, liquid expanded, as the lateral surface pressure is reduced and the area per lipid molecule is increased). (see Phillips, M.C., et al., Biochimica et Biophysica Acta, 163: 301-313 (1968)). In comparison, albumin shells tend to be rigid, and, for the most part, are often incapable of greater than 5 percent expansion. The expansion of the shells may be an important performance parameter in certain applications. For example, as microbubbles subjected to ultrasound particles are believed to undergo cycles of expansion and contraction.
The employment of phospholipid is also advantageous since it is generally biocompatible. Although deposition of albumin to a surface can passivate the surface to certain types of adhesion, denatured albumin on a surface may possibly act as a specific ligand for MAC-1, a receptor on white blood cells. Shafer et al. propose that denatured albumin nano- and micro-spheres incubated with macrophages were phagocytosed within two hours (see Shafer, V. et al., Journal of Microencapsulation 11(3): 261-269 (1994)). The "stealthy" phospholipid vesicles composed of a lipid/PEG-lipid mixture has been shown to evade the reticuloendothelial system and exhibit prolonged blood circulation times (e.g., half-life of five hours) relative to non-PEGylated vesicles (half-life of less than 30 minutes) (see, Klibanov, et al., FEBS Letters, 268(1): 235-237 (1990)). Thus, depending on the system and the application, the ability of albumin-coated microbubbles to stimulate macrophages and neutrophils may or may not be advantageous. With the lipid-coated microbubbles though, the ability to express the PEG coating with specific molecular ligands for macrophages or neutrophilis as desired is believed to be advantageous over the simple albumin microsphere, and allows these interactions to be controlled, (elicited or suppressed depending on the application).
Notwithstanding the above, there remains a need in the art for methods of forming gas microbubbles with lipid-containing shells that allow for one to better control the physical properties of the shells in the microbubbles such that they are suitable for various applications.